Recently we have over-expressed the enzyme a 1,6-fucosyltransferase from Rhizobium sp. in Escherichia coli. In this heterologous system the enzyme was mainly expressed as inclusion bodies and the one that was expressed soluble showed a shortlasting activity in solution due to precipitation of the protein. A structural analysis of the sequence using the TMpred program
predicted a highly hydrophobic region of 19 aa close to the C-terminal of the protein. In order to investigate the influence of this region on the formation of inclusion bodies and the precipitation from solution, we cloned a truncated version of the protein where a C-terminal fragment of 65 aa, including the predicted transmembrane-like region, was removed. The resulting protein was expressed in a soluble form without formation of inclusion bodies. The truncated protein catalyzed the transfer of a fucopyranosyl moiety from GDP-b-l-Fucose to chitobiose. Comparison of the acceptor specificity between the truncated a 1,6-fucosyltransferase and the wild-type enzyme, showed a similar behavior for both enzymes. Our results indicate that the active center is not located in the C-terminal extreme of the protein in contrast to the case of the mammalian glycosyltransferases. Also, these results indicate that the a-6-motif III is not directly involved in the catalytic activity of the enzyme.

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This work was supported by the Spanish DGES (Grant PB96-0828) and Comunidad de Madrid (Grant 07B/0027/1999).

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458854 bytes

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application/pdf

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eng

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Pergamon Press

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openAccess

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1,6-Fucosyltransferase

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Rhizobium Sp.

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Activity of the enzyme

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C-terminal

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protein

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TMpred

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C-Terminal Truncation of α 1,6-Fucosyltransferase from Rhizobium Sp. does not Annul the Transferase Activity of the Enzyme